CROSS-REFERENCE TO RELATED APPLICATIONS
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This application is a continuation of U.S. patent application Ser. No. 10/912,405, filed Aug. 5, 2004, which was a continuation-in-part of U.S. patent application Ser. No. 09/938,131, filed on Aug. 23, 2001, which claimed the benefit of U.S. Provisional Patent Application Ser. No. 60/227,254, filed Aug. 23, 2000.
FIELD OF THE INVENTION
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The ability to detect anti-HCV in oral fluid is useful for the rapid detection of HCV exposure by non-invasive means. The methods provided in the invention are also useful in the early detection of HCV infection by recognition of anti-HCV of the IgA class, monitoring of antiviral therapy, genotyping of HCV virus, determining immune response to individual HCV epitopes, and monitoring potential vaccination programs.
BACKGROUND OF THE INVENTION
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Hepatitis C(HCV) is the major cause of parenterally transmitted non-A, non-B hepatitis (Choo et al., 1989 Science 244:359-362; Kuo et al., 1989, Science 244:362-364) with a prevalence of 1-3% throughout the world (Davis et al., 1998, Hepatology 28(Suppl 4, pt 2):99A). Chronic disease develops in 60-85% of patients, with cirrhosis representing a major hallmark of HCV infection. Among patients whose infection progresses to cirrhosis, as many as 1-4% develop hepatocellular carcinomas annually (Fattovich et al., 1997, Gastroenterology 112:463-472). It is estimated that the need for hepatic transplantation for infected individuals will increase 5-7 fold in the next 20 years unless more effective treatments and preventative programs are introduced (Davis et al., 1998, Hepatology 28 (Suppl 4, pt 2):99A).
While additional anti-viral therapies are needed to combat the spread of HCV, equally necessary is the development of a rapid, highly sensitive and cost-effective test to detect and monitor HCV within the population. Current PCR and ELISA-based assays for the detection of HCV are costly, relatively slow and reliant upon serum or plasma as the sample fluid. The substitution of oral fluid for serum in HCV assays would provide a cost-effective, non-invasive means to conduct routine screening and would facilitate sample procurement from patient groups where serum collection is difficult, such as intravenous drug users, who constitute a significant portion of total HCV cases.
A number of oral fluid-based assays have been designed for the detection of viral antibodies with good results. Virus-specific antibodies have been detected in the oral fluid of patients infected with human immunodeficiency virus (Major et al., 1991, J. Infect. Dis. 163:699-702), hepatitis A (Stuart et al., 1992, Epdiem. Infect. 109:161-166), hepatitis B (Ben Aryeh et al., 1985, Arch. Oral Biol. 30:97-99), rubella (Saleh, 1991, J. Egypt Public Health Assoc. 66:123-124) and following immunization against polio (Zaman et al., 1991, Acta Paediatrica Scan. 80: 1166-1173), rotavirus (Ward et al., 1992, J. Med. Virol. 36: 222-225) and hepatitis A (Laufer et al., 1995, Clin. Infect. Dis. 20:868-871). For HCV, ELISA-based assays developed initially for use with serum or plasma have been modified to detect anti-HCV antibodies in oral fluid (Cameron et al., 1999, J. Viral Hepatitis 6:141-144; Elsana et al., 1998, J. Med. Virol 55:24-27; McIntyre et al., 1996, Eur. J. Clin. Microbiol. Infect. Dis. 15:882-884; Sherman et al., 1994, Amer. J. Gastroent 89:2025-2027; Thieme et al., 1992, J. Clin. Microbiol. 30:1076-1079); using a modified protocol with the HCV 3.0 ELISA (Ortho Diagnostic Systems), (McIntyre et al. 1996, Eur. J. Clin. Microbiol. Infect. Dis. 15:882-884) detected anti-HCV antibodies within a group of 18 HCV(+) and 49 HCV(−) oral fluid samples with 72% sensitivity and 98% specificity. In the same study, 100% sensitivity and 100% specificity was achieved using the Monolisa HCV assay (Sanofi Pasteur Diagnostics, France). It is unclear what the differences were that lead to the increased sensitivity of the Monolisa test, and thus care must be taken in the interpretation of results obtained from tests not designed specifically for use with oral fluid. None of these assays has achieved the sensitivity required for a rapid point of care test. None of these assays has disclosed the special role of oral fluid IgA in human oral fluid as a key determinant of sensitivity and specificity for HCV screening.
An intrinsic difficulty in designing oral fluid-based diagnostic assays, however, is detecting a sufficient proportion of the relatively low levels of antibody present in oral fluid to generate a meaningful diagnostic result. Indeed, it is estimated that overall antibody levels are 800-1000-fold lower in oral fluid than in serum (Parry et al., 1987, Lancet 2:72-75) making detection sensitivity of the utmost importance in oral fluid-based tests. While this problem is significant, an HCV assay designed to be used specifically with oral fluid as the diagnostic fluid, and not simply a serum-based assay modified for use with saliva, could overcome this complication and provide an important test for HCV in the population.
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OF THE INVENTION
The invention disclosed is a means to detect antibodies against HCV using oral fluid as a sample medium. Assays in the prior art have not achieved the sensitivity and specificity required to rapidly screen HCV infection in human oral fluid. Most critically, the use of a labeled detection molecule that recognizes not only IgG, but all classes of immunoglobulins, enhances the ability to detect anti-HCV in oral fluid in an ELISA format or using a flow-through system. When detecting anti-HCV using a labeled detection molecule that recognizes only anti-HCV of the IgG class, detection sensitivity was vastly reduced. The incorporation of a detection method that labels multiple classes of anti-HCV, on the other hand, allows for increased detection sensitivity of samples that would otherwise be scored negative using a detection method that only recognizes IgG.
By coupling this detection method to an assay that utilizes a membrane with immobilized HCV peptide antigens present as a trapping zone, followed by subsequent flow of sample through the trapping zones and selective binding of labeled antibodies specific for HCV epitopes within the trapping zone, an immunoassay for the detection of anti-HCV can be performed in a short time period (<15 minutes). The ability to use oral fluid as a sample is of great value to such a rapid diagnostic tool since oral fluid can be collected rapidly and used immediately following collection. An assay using oral fluid, performed on a miniature test platform, analyzed in a small light gathering machine, and able to be completed within 15 minutes from start to finish would be of enormous value as a screening agent for HCV in the population. By decreasing the time of the assay and eliminating the need for invasive blood-based sample acquisition, such an assay would certainly increase the ability to screen, detect and monitor HCV within the population.
The use of an assay to detect anti-HCV in saliva would also be of benefit in the rapid and non-invasive detection of antibodies following vaccinations and monitoring of vaccination efficacy over time, monitoring therapeutic response of patients to treatment regimes and screening for early infection, as IgA antibodies are known to be an important part of the early stages of the immune response.
Thus, the present invention seeks to overcome the deficiencies of the prior technology by designing an HCV assay that would meet the following objectives. A first objective is that the test is non-invasive, generates minimal risk of infection to those administering the test and can be performed from start to finish by non-medical personnel.
A second objective is that the test is rapid (<15 min.).
A third objective is that the test is specialized to detect the specific class of anti-HCV antibodies in oral fluid, and is not simply a modification of a current serum-based assay.
A fourth objective is that the test incorporates a number of different HCV antigens to minimize false negative results.
A fifth objective is that the test is adaptable to future incarnations of the assay to meet specific diagnostic needs, and that it is sensitive enough to detect extremely low levels of anti-HCV.
BRIEF DESCRIPTION OF THE DRAWINGS
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FIG. 1. Characterization of multiple classes of anti-HCV present in serum and oral fluid. Paired serum/oral fluid samples were screened by HCV 3.0 ELISA using enzyme-conjugated antibodies specific for human IgG, IgM or IgA, respectively. (A) In serum, high levels of anti-HCV IgG and IgM class antibodies are detectable, while relatively little anti-HCV IgA is present. (B) In oral fluid, the majority of antibodies detectable are of the IgG or IgA class with little or no anti-HCV IgM present.
FIG. 2: Components of the HCV strip immunoassay. (A) Top view of disassembled assay cassette showing the position of the nitrocellulose test strip as well as the top and bottom wicks and the substrate-coated gelbond. The “trapping zone” is located directly beneath the substrate-coated gelbond. The trapping zone and substrate-coated gelbond are kept from contacting one another until such time as the cassette is inserted into the luminometer for reading. (B) Top view of an assembled cassette with the conjugate hinge in the open position. (C) Top view of an assembled cassette with the conjugate hinge in the closed position. Also visible in C is the chase injection port and the luminescence measuring window. (D) Side view of assembled cassette showing the conjugate hinge in the open position as well as the lever on the back of the cassette that is contacted by the Junior luminometer upon insertion to bring the anti-HCV/anti-human-AP complex captured in the trapping zone into contact with the substrate-coated gelbond suspended above and thus initiate the luminescence reaction.
FIG. 3: Dose response curve for spiked monoclonal anti-HCV antibodies in an HCV(−) oral fluid sample. Monoclonal anti-HCV antibodies were spiked into an HCV(−) oral fluid sample to test the ability of the mixed antigen trap to capture anti-HCV antibodies. (A) Dose response curve for spiked monoclonal anti-HCV antibodies present in oral fluid at concentrations ranging from 0-117 μg/ml. (B) Digital photograph of nitrocellulose test strips post-stained with NBT/BCIPT corresponding to the data points in (A). Staining within coherent trapping zones is visible in all spiked samples while no staining is present in the non-spiked control.
FIG. 4. Screening of 64 known HCV(+) saliva samples on strip immunoassay. A cutoff was determined using the mean of 14 HCV(−) saliva samples+2SD. 63/64 known HCV(+) saliva samples generated values above the calculated cutoff for a sensitivity of 98.4%.
FIG. 5: Direct visualization of HCV LNSI assays using the NightOwl Molecular Light Imager. To observe the relative amounts of luminescence produced by highly immunoreactive oral fluid samples (HCV(++)), weakly reactive samples (HCV(+)) and HCV(−) samples, luminescence was collected (60 sec exposure time) by the NightOwl Molecular Light Imager. A luminescence intensity scale is provided for reference with purple representing the most intense regions of luminescence. (A) A highly immunoreactive oral fluid sample generated extensive luminescence within the collection window just below the closed conjugate hinge assembly. (B) A weakly immunoreactive oral fluid sample. (C) An HCV(−) oral fluid sample.
FIG. 6. Antibody profile of 9 individual patient samples using a six-line peptide trapping zone. Patient samples were passed through six different antigen trapping zones to observe the immune response profile for these subjects. Strong responders had high levels of antibody binding against most of the six peptides while weak responders had lower levels of binding within the six trapping zones.
FIG. 7 Diagram of the steps of the process according to one embodiment of the invention.
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OF THE INVENTION
The invention described herein represents the ability to detect HCV exposure in oral fluid by labeling and detecting multiple classes of anti-HCV instead of anti-HCV IgG alone. Saliva is first collected by a device independent of the test module. A volume of crude saliva is then added to the test module wherein it mixes with a detection molecule that labels all classes of human antibodies. The antibody-detection molecule complex then passes through a trapping zone comprised of immobilized HCV peptide antigens. Antibody/detection molecule complexes that are recognize the HCV sequences represented in trapping zone bind and are thus immobilized within the zone (FIG. 7). The addition of a suitable substrate for the detection molecule allows for generation of a signal in samples possessing antibodies to HCV and thus correlates with HCV exposure. In a particular embodiment the non-antibody label protein is protein LA conjugated to an enzyme which generates a chemiluminescent signal that is read in a luminometer.
I. Detection of Multiple Classes of Anti-HCV in Oral Fluid to Increase Detection Sensitivity.
The detection of multiple classes of anti-HCV in oral fluid can increase the detection sensitivity of the Ortho HCV 3.0 ELISA to levels comparable with those attained using serum samples. Patients for this study were pre-selected from one of eleven participating clinical sites and shown to be either HCV positive or negative based on a clinical diagnosis according to the CDC testing algorithm (Alter et al., 1998). Serum samples were further confirmed by repeat in-house testing using the Ortho HCV 3.0 ELISA following the manufacturers instructions. Oral fluid samples were collected using a Salivette (Sarstedt Research, Germany) whereby a polyester-coated cotton plug is placed in the mouth of the patient until saturation and is then centrifuged in a carrier tube for 5 minutes to extract the oral fluid. The Salivette was chosen for its ease of use and because it does not use a sample buffer to dilute the specimens. Paired samples were shipped overnight at 4° C. and processed immediately upon arrival. Samples were then stored at −80° C. until testing.
To determine if specific classes of antibodies were preferentially enriched in serum or oral fluid samples, the composition of anti-HCV present in both fluids was examined. Fourteen paired HCV-positive oral fluid/serum samples (with sufficient volumes of oral fluid for multiple ELISA assays) were chosen for ELISA analysis and examined using secondary enzyme-conjugated antibodies (Jackson Immunoresearch) that recognize only IgG, IgM or IgA, respectively, to identify the different classes of anti-HCV detectable in oral fluid (FIG. 1). Modification of the HCV 3.0 was necessary to achieve optimal detection sensitivity and specificity; compared to the manufacturers instructions for use with serum, oral fluid sample volume was increased from 10 μl to 100 μl per well and sample incubation time was increased from 1 hour at 37° C. to overnight at 4° C. Furthermore, a more sensitive two-part TMB substrate kit (Pierce) was used for all testing in place of the o-phenylenediamine tablets supplied with the HCV 3.0 kit. Analysis of the optical densities (OD) generated by these 14 samples showed that anti-HCV of the IgG and IgM class was most abundant in serum samples (mean OD=1.85, 1.03, respectively), with little IgA class anti-HCV present (OD=0.24; FIG. 1A). These samples were not treated for rheumatoid factor, however, and thus it is possible that elevated levels of anti-IgM reactivity in serum samples may be attributable to the presence of this interfering substance (see Genser et al., 2001). In contrast, while IgG (OD=1.10) remained the major class of anti-HCV detectable in oral fluid samples using the HCV 3.0 assay, a higher level of anti-HCV IgA (OD=0.42) was also detectable, while nearly no anti-HCV IgM was present (OD=0.02; FIG. 1B). Statistically, the mean OD of anti-HCV of the IgG and IgM class is significantly reduced in oral fluid compared to serum (P<0.01), while the OD of IgA class anti-HCV is not significantly different (P>0.01).
Unexpectedly, in a number of oral fluid samples possessing low anti-HCV IgG levels, a significant amount of anti-HCV IgA was detectable (FIG. 1B) which might contribute to a higher overall OD and thus render a positive result. Indeed, the ability to detect anti-HCV of the IgA class may also increase the likelihood of detection early on during the course of infection, as IgA is known to be present during the earliest stages of the immune responses to infections. (Freihorst and Ogra, 2001).
An investigation was then conducted to determine whether the detection of multiple classes of anti-HCV antibodies, instead of IgG alone, could increase the sensitivity of the Ortho HCV 3.0 ELISA in a modified oral fluid-based format. Paired oral fluid/serum samples from 127 known HCV seropositive and 31 seronegative donors were screened using the HCV 3.0 assay according to the manufacturer's instructions using the monoclonal anti-human IgG-peroxidase detection antibody. Using serum samples, 100% sensitivity and specificity with the HCV 3.0 assay was achieved (Table I).
Sensitivity and specificity of HCV 3.0 assay using paired serum/oral
fluid samples with different enzyme-conjugated secondary antibodies.